This invention relates generally to a method for performing flow field-flow fractionation (flow FFF), which is useful in the separation, isolation, and characterization of a wide range of particles and macromolecules.
There is a tremendous need in virtually all branches of science and technology for the separation and characterization of a wide variety of analytes. Although numerous methods have been devised to accomplish such separations, including various forms of chromatography, filtration, precipitation, electrophoresis, and centrifugation, no one technique is universally applicable. One useful family of techniques is field-flow fractionation (FFF), as taught by J. Giddings in U.S. Pat. No. 3,449,938 and recently reviewed by P. Schettler, LC-GC 14(10), 852 (1996), which are incorporated by reference herein. The most universally applicable of these FFF techniques is flow field-flow fractionation (flow FFF), as taught by J. Giddings in U.S. Pat. No. 4,147,621, which is incorporated by reference herein. Flow FFF devices effect the fractionation of particles by pushing an analyte contained in the xe2x80x9cchannel flowxe2x80x9d stream (symbolized herein as xe2x80x98Vzxe2x80x99) axially along the surface of a filtration membrane inside a narrow channel while simultaneously pushing a xe2x80x9ccross flowxe2x80x9d stream (symbolized herein as xe2x80x98Vxxe2x80x99) through the channel in a direction orthogonal to the channel flow stream Vz. Inside the channel, these two flowstreams intersect and intermingle, and the crossflow stream Vx provides a field of hydraulic force across the planar surface of the filtration membrane which is sufficient to permit the separation of analyte samples into their constituent components based on differences in their hydrodynamic sizes or diffusion coefficients. The crossflow stream thus provides a force field which results in the retention of the analyte. Larger analytes feel or sense the force of this crossflow stream more strongly, due to their larger Stokes radii, and therefore spend their time, on average, closer to the filtration membrane. Smaller analytes feel the crossflow stream more weakly, and diffuse away from the filtration membrane to occupy a higher average position over it, where they encounter the faster flowstreams of the Poiseuille flow velocity distribution under laminar channel flow conditions and are carried along the channel faster and elute as a peak sooner than larger analytes. In this way, the analyte particles are fractionated as a function of their diffusion coefficients or apparent hydrodynamic sizes.
Many publications, such as J. Giddings et al., Polym. Mater. Sci. Eng. 65, 21 (1991), P. Schettler, LC-GC 14(10), 852 (1996), J. Giddings, Sep. Sci. Technol. 24(9and10), 755 (1989), J. Giddings et al., Meth. Biochem. Anal. 26, 79 (1980), F. Yang et al., Anal. Chem. 49(4), 659 (1977), and M.-K. Liu et al., Anal. Chem. 63, 2115 (1991), and a number of patents, such as U.S. Pat. Nos. 4,214,981, 4,737,268, 4,830,756, 4,894,146, 5,039,426, 5,141,651, and 5,193,688, all of which are incorporated by reference herein, have disclosed applications, modifications, calibration procedures, and improvements to the flow FFF separation process, so that the set of conditions which are classically employed in flow FFF systems is well known and fully illustrated in the prior art.
One important advantage of flow FFF is the number and variety of the variables which are available in the design of channels and the operating parameters. For example, although thin, planar channels are the most common, an annular channel geometry is also known, as illustrated in FIG. 7 of U.S. Pat. No. 4,214,981. In addition, at least three different xe2x80x9coperating modesxe2x80x9d are known for flow FFF, including the xe2x80x9cnormal,xe2x80x9d xe2x80x9csteric,xe2x80x9d and xe2x80x9chyperlayerxe2x80x9d modes. In the normal mode, the analyte exists predominantly as a xe2x80x9ccloudxe2x80x9d of particles hovering over the surface of the membrane, balanced between the crossflow force driving it towards the accumulation wall on the one hand and diffusion, flow-induced lift forces, and steric effects on the other hand, driving the analyte away from the accumulation wall during its travel down the channel. In this normal mode, which takes place with small particles at modest flow rates, the contribution of flow-induced lift forces is considered to be minimal, so that the primary forces acting on the particles are the hydraulic crossflow force field and the back-diffusion of the analyte away from the filtration membrane. Thus, in normal mode, smaller particles elute before larger particles. In the steric mode, which predominates for larger particles and relatively high crossflow velocities, the particles essentially reside along the surface of the filtration membrane, so that larger analyte particles protrude further into the channel and sample the faster flowstreams of the Poiseuille flow velocity distribution. Thus, in steric mode the particles are considered to essentially xe2x80x9crollxe2x80x9d along the surface of the filtration membrane, so that the elution order is the reverse of that observed for normal mode, with larger particles eluting before smaller ones. In hyperlayer mode, which takes place with similar larger particles as required for the steric mode but with somewhat faster flow velocities, the flow-induced lift forces which are considered to be minimal in normal mode become significant, and lead to a fluid force lifting the particles off from the surface of the membrane and into a relatively narrow layer of fluid. As with steric mode, larger particles in hyperlayer mode elute before smaller particles.
In addition, there also exist at least two configurations of flow FFF channel. In xe2x80x9csymmetricalxe2x80x9d flow FFF configuration, the crossflow stream Vx enters the channel from the crossflow inlet frit above it and passes through the channel and then through the filtration membrane, whereupon retained analyte is removed from the cross flow and the remaining Vx stream exits through the outlet frit underneath the filtration membrane. In symmetrical flow FFF, the channel flow stream Vz is thus physically distinct from the cross flow stream Vx, and these two flow streams are typically xe2x80x9cbalancedxe2x80x9d by independently adjusting the four flow streams Vzin, Vzout, Vxin, and Vxout by any convenient means until Vzin equals Vzout and Vxin equals Vxout. However, these two flow streams need not be balanced in this manner, since the only physical requirement is that the total of the sum of Vzin and Vxin is equal to the total of the sum of Vzout and Vxout. Thus, symmetrical flow FFF channels can also be operated in xe2x80x9cunbalanced flowxe2x80x9d mode, in which the relative velocities of the four flow flowstreams can be adjusted to obtain any desired effect. In xe2x80x9casymmetricalxe2x80x9d flow FFF, the crossflow inlet frit is replaced with a non-porous solid material, so that the channel flow stream Vzin and the crossflow stream Vxin must be mixed and pumped into the channel together through a single channel flow inlet. Typically, the total input flux is chosen in advance, and the ratio of the fluid flow which exits from the channel flow outlet to that which exits through the filtration membrane and the crossflow outlet can be controlled by any convenient means, including pieces of constrictive tubing, pressure regulators, etc. In this way, the desired degree of retention can be achieved.
The flow FFF channel can also contain one of a number of potential channel spacer xe2x80x9cgeometries,xe2x80x9d typically produced by cutting a portion out from the center of a thin sheet of spacer material, most often a plastic. For example, if a portion of the spacer consisting of a rectangle 2.0 cm wide by 25 cm long and bearing end-pieces consisting of isosceles triangles 2.0 cm on a side is removed, and the apparatus assembled, then the channel would exhibit a xe2x80x9cparallel-walledxe2x80x9d geometry. Similarly, a triangular portion of 2.0 cm breadth and 20 cm length contiguous to an isosceles triangles portion 2.0 cm on a side would yield a geometry referred to as xe2x80x9ctapered-wall.xe2x80x9d Although a large number of such geometries are possible, the parallel-walled and tapered-wall, in combination with various end-piece geometries, are the most common.
These two possible channel forms, three modes of flow FFF, two types of flow FFF channel, and large number of potential channel geometries are all independent and non-exclusive, and can exist in almost any combination. For example, a thin planar channel form with an inlet frit operated at modest flow rates with small analyte particles can be described as a planar symmetrical normal-mode parallel-walled separation, while an annular channel form with a solid internal core, a single, combined channel flow and crossflow inlet, and a filtration membrane supported by the outside frit operated at high flow rates with large analyte particles can be described as an annular asymmetrical steric-mode separation. All of these potential combinations are apparent to those skilled in the art.
Although it is in principle applicable to a wide variety of analytes, flow FFF has met with only modest commercial success, in comparison with related techniques such as size-exclusion chromatography (SEC). This lack of success is due primarily to the inherent limitations of the existing methodology and instrumentation, which include strong and unpredictable interactions with membranes, poor resolution, and especially the peculiar requirements resulting from the need for relaxing the analyte sample prior to commencing the fractionation, as described more fully below.
Flow FFF separations both cause and require the analyte to be in an equilibrium position against the filtration membrane, balanced between the crossflow force driving it towards the filtration membrane on the one hand and diffusion, flow-induced lift forces, and steric effects on the other hand, driving the analyte away from the filtration membrane during its travel down the channel. However, when the analyte sample is first introduced into the channel, it is distributed essentially evenly over the entire incoming cross-sectional area, far from the desired equilibrium distribution against the filtration membrane, as described more fully in M.-K. Liu et al., Anal. Chem. 63, 2115 (1991). After introduction of the analyte sample into the channel, the analyte will relax to its equilibrium distribution close to the filtration membrane under the influence of the crossflow field which drives the flow FFF separation. In order to accomplish a flow FFF separation of reasonable resolution, the relaxation of this incoming analyte must be occur before it is pushed any significant distance axially along the membrane""s surface by the channel flow. That is, continuing the channel flow during this period of crossflow relaxation leads to unacceptably broadened analyte peaks, since the widely-varying flowrates of the different laminae across the incoming cross-sectional area will deposit the analyte in a very broad pattern onto the filtration membrane.
Although it has long been presumed (see e.g. J. Giddings et al., Meth. Biochem. Anal. 26, 79 (1980)) that a sufficiently large field, with its resulting increased retention of the analyte, combined with a decreased channel thickness and a low channel flow velocity would attenuate this relaxational broadening, it has never been possible to make the relaxation effect negligible in flow FFF. For this reason, as described for example by F. Yang et al., Anal. Chem. 49, 659 (1977), and by Liu et al. in Anal. Chem. 63, 2115 (1991), as well as in other references, the standard procedure in flow FFF has long been to stop the channel flow for a period of time which is sufficient to relax the analyte to its equilibrium position against the membrane, before continuing the flow FFF experiment. In this so-called xe2x80x9cstop-flowxe2x80x9d relaxation procedure, the analyte enters the channel via the axial or channel flowstream for a period of time which is estimated to be sufficient to remove all of the analyte from the inlet tubing and deposit it onto the channel. The channel flow (Vz) is then stopped in order to control band distortion and broadening, typically by moving a flow-switching valve, and the cross flow (Vx) is continued for a period of time which is estimated to be sufficient to establish the analyte in its equilibrium position with respect to the filtration membrane. This period of stopped flow is typically sufficient to allow 1-2 times the channel volume to pass through the channel in the cross flow, and usually requires between 5 seconds and several minutes (see, for example, the stop-flow time (tsf) of 120 seconds required for DNA in FIG. 5 of J. Giddings et al., Polym. Mater. Sci. Eng. 65, 21 (1991)). After the stop-flow period, the axial flow is re-established, typically by returning the flow-switching valve to its original position, and the analyte is eluted from the channel. Thus, during this stop-flow relaxation process, analyte components which are distributed widely in the flowstream laminae entering the channel are forced into a narrow cross-sectional region close to the filtration membrane from which a reasonably efficient separation can take place. Failure to fully relax the sample prior to the fractionation experiment is known to result in a large degree of dispersive broadening of the analyte, leading to unacceptably poor resolution and preventing the sample components from being distinguished from one another.
Unfortunately, even in the best of cases, this so-called stop-flow procedure leads to flow instabilities and other problems which are manifest as baseline shifts and extraneous peaks. For example, one common problem with stop-flow relaxation is the presence of so-called xe2x80x9cvoid peaks,xe2x80x9d which elute at or near the geometric void volume of the channel (see, for example, the large void peak in FIG. 2 of J. Giddings et al., Polym. Mater. Sci. Eng. 65, 21 (1991)). Furthermore, the stoppage of axial hydrodynamic motion over the surface of the membrane frequently leads to particle adhesion to the channel walls, and especially to the filtration membrane. The stop-flow procedure also increases the run time by at least the stop-flow period, and axial dispersion continues to take place during this stop-flow period, so that the peak continues to broaden while the relaxation is taking place. The hydraulic movements inside the channel during the stopping and restarting of the channel flow cause further disruption of the analyte zone, leading to additional band spreading and a loss of resolution. In addition, the stop-flow procedure renders very difficult the use of certain detectors, such as light-scattering (LS) and refractive-index (RI) detectors, which are sensitive to the pressure changes which invariably accompany flow-stream switching. In the case of LS detectors, the stop-flow procedure itself causes additional artifactual peaks to be detected, which appear to result at least in part from the removal of particles from the membrane and channel surfaces by the resulting hydraulic shock wave. Stop-flow relaxation also requires additional switching valves, tubing, and other components, as well as controlling software and signal pulses for the activation of the additional components, rendering the process more complicated and expensive. Thus, methods which require the stop-flow relaxation procedure result in excessive broadening of analyte peaks, compromise the effectiveness and utility of detectors which are among the most useful methods for characterizing fractionated species, and overly complicate and encumber the flow FFF process while increasing the cost.
Despite the many disadvantages of the stop-flow procedure, however, the system void peaks are useful in determining the void time t0 and the void volume V0 of the channel. The retention time tr of the true void peak (normally the second of the two early-eluting system peaks) is converted into a retention volume using the known channel flow rate Vz. From this volume is subtracted the total void volumes of the components immediately preceding the channel, including the injector, any valves, and the tubing. The difference yields the true void volume V0 of the channel, which can be converted back to the true void time t0 by dividing it by the channel flow rate Vz. These true void times and void volumes are useful in calculating the channel width w, from which many other parameters useful in the characterization of both the analyte sample, including the distribution of diffusion coefficients or frictional coefficients, and of the separation, including measures of the efficiency such as the height equivalent to a theoretical plate, the resolution, the fractionating power, and the selectivity, can be computed. To be most useful, any improved method which could eliminate the early-eluting system peaks observed with the stop-flow relaxation procedure should also be accompanied by a method for calculating either the channel void time t0, the void volume V0, or the channel width w which does not depend on identifying a discrete void peak in the fractogram.
The severe limitations of the stop-flow technique have long been recognized, and another technique, hydrodynamic relaxation, has resulted from attempts to avoid these pitfalls. Two variants of this technique, inlet splitting and frit inlets, are described more fully in M.-K. Liu et al., Anal. Chem. 63, 2115 (1991). In each of these methods, the analyte is driven close to its equilibrium position by a higher-velocity flow substream rather than by the lower-velocity crossflow field in the channel. In the frit-inlet method, disclosed more fully by Giddings in U.S. Pat. No. 5,193,688 and described in M.-K. Liu et al., Prot. Sci. 2, 1520 (1993), hydrodynamic relaxation is carried out by the introduction of an additional flowstream into the channel through an additional inlet frit near the entrance of the main axial flow bearing the analyte. This so-called xe2x80x9cfrit-inlet flowxe2x80x9d enters the channel from a position near the inlet cross flow frit and, by virtue of its volume, pushes the existing contents (which enter the channel through the main axial entrance) towards the accumulation wall. In effect, this xe2x80x9crelaxesxe2x80x9d the analyte towards the membrane at a rate which is faster than the velocity of the crossflow field. Although this technique can work reasonably well for certain analytes, it has at least six important disadvantages. First, it is not generally applicable, and second, it often results in a loss of resolution as compared with the stop-flow relaxation method. Third, it requires the use of a separate flowstream, necessitating an additional pump (this is typically the third pump in the system). Fourth, the increased effective cross flow field under the inlet frit leads to increased and complicated adsorption of the analyte to the underlying membrane, requiring in the best case extensive calibration of the channel for each analyte and set of conditions. Fifth, frit inlet relaxation is known to cause artifactual aggregation of some analytes. Finally, the frit-inlet method also adds a large volume of solvent to the analyte, diluting it and rendering its subsequent detection and characterization more difficult.
In an effort to overcome the dilution of the analyte by an inlet frit, or to concentrate a dilute analyte as it emerges from the channel, an outlet frit can be added which is capable of stripping off the bulk liquid flowing above the sample layers in the channel. This outlet frit effectively concentrates the analyte components, aiding in their detection, characterization, or collection. Outlet frits suffer from some of the same problems which plague inlet frits, including a loss of resolution, and also introduce yet another flow stream, confounding the process of balancing the flow rates and unduly complicating the flow FFF procedure.
The latter difficulty can be mitigated somewhat by combining the outlet frit with an inlet frit to produce a channel architecture referred to as a xe2x80x98frit inlet/frit outletxe2x80x99 system. If these two flow streams are independent, at least one additional pump is required for the inlet frit, although two additional pumps are sometimes used, and this flow stream also has to be taken into account when balancing the flow rates. The flowstreams from these two frits can be closed into a circle by routing the output of the outlet frit into the intake of a pump which then pumps the fluid into the inlet frit, somewhat simplifying the fluidic configuration, since the flowrates into and out of the respective frits are known and fixed. However, such a system still requires at a minimum one additional pump, and this circular pumping scheme also imposes impossible demands on the performance of that pump. The pump must produce smooth, pulseless flow out into the channel to preserve the laminar fluid flow required for flow FFF separations, and must also take the split frit out flow smoothly into its intake manifold, again without pressure fluctuations. At the present time, there is no commercially-available pump which completely fulfills these requirements, so that the use of the split inlet/outlet configuration must be confined to systems where low resolution and the use of only relatively pressure-insensitive (e.g. UV) detectors are acceptable. Specifically, frit inlet/frit outlet systems cannot be used quantitatively with LS detectors, and also cause RI detectors to perform poorly. Furthermore, outlet frits inevitably remove some small fraction of the analyte, and re-introducing this material into the inlet frit can lead to significant contamination of the incoming analyte and a reduction in the efficiency and resolution of the fractionation. Thus, frit inlet/frit outlet systems add undue difficulty and cost to the fractionation while also decreasing its resolution and efficiency and suffering from all of the deficiencies of frit inlet and frit outlet systems mentioned above.
In the alternative hydrodynamic relaxation technique, the inlet-splitter method, relaxation is carried out by the insertion of a thin flow splitter to divide the inlet region into two slit-like flow spaces. Manipulation of the flow rates of the incoming streams entering above and below the splitter, so as to obtain a neat carrier flow rate which is significantly greater than the flow rate of the sample-bearing stream, drives the latter into a thin laminus close to the accumulation wall of the channel, where it is close to its equilibrium position against the accumulation wall. However, there are several disadvantages to the use of such inlet splitters in flow FFF systems. First, of course, is the introduction of yet another flow stream. Second, for proper operation the inlet splitter must be suspended evenly across the several-centimeter-wide gap of the thin channel; unevenness of even a few tens of micrometers will noticeably distort the hydrodynamic relaxation process. A third difficulty is that the introduction of a flow splitter and the two associated flow spaces on either side of the splitter, yielding a total of three layers in all, is very often inconsistent with the utilization of very thin, high-performance FFF channels (e.g. channel widths of roughly 50-508 xcexcm), Fourth, since the flow stream in which the sample is introduced must traverse the narrow gap on one side of the splitter, where the thickness is only a fraction of the full channel width, there is an enhanced risk that larger particles in the sample, whether part of the sample or an impurity therein, will clog all or part of the fluid path needed for sample introduction. Fifth, at high flowrates the abrupt change in flow direction at the splitter edges may introduce eddy currents in the fluid capable of disrupting the distribution of components near the inlet and outlet. Furthermore, this technique does not exhibit the same high resolution as other flow FFF relaxation methods, so that such split-inlet cells have been largely confined to continuous, binary separations (for example, the SPLITT channel illustrated in FIG. 2 of P. Schettler, LC-GC 14(10), 852 (1996)).
A fourth type of sample relaxation, reversed-flow focussing, is used almost exclusively in asymmetrical flow FFF due to the inability in this mode to independently control the channel and crossflow rates. It is, however, also compatible with the operation of symmetrical flow FFF channels. In reversed-flow focussing not only is the axial flow interrupted for a period of time, but the flow direction is actually reversed during the relaxation period. In order to accomplish this, the reversed flow passes backwards through at least the Vz outlet from the channel, and the flow may even be reversed through one or more of the detectors. The two flows, entering from opposite ends of the channel, meet at a point determined by the relative volumetric flow rates of the two streams, whereupon all of the flow passes through the membrane and out of the channel through the crossflow outlet frit. Such a scheme is not possible for many fluorescence and RI detectors, which cannot tolerate back-pressure on their delicate flow cells, and also is highly disruptive for LS detectors, which are quite difficult to use quantitatively in this way. However, even if the LS and RI detectors are bypassed in the reversed-flow pathway, then the interdetector broadening can become unacceptably large, due in part to the required flow-switching valve between the channel and the detectors, complicating accurate characterization of the analyte. Bypassing the LS and RI detectors also leaves no solvent flowing through them during the relaxation period, requiring yet another pump to keep fluid flowing through them at precisely the same flow rate and pressure to minimize the baseline disturbances which result from the hydraulic changes in the channel. Furthermore, the reversed-flow focussing procedure is known to cause artifactual aggregation of many important analyte species, including proteins, polymers, and liposomes. This results largely from the fact that the two opposing flowstreams tend to concentrate the analyte against the filtration membrane in a relatively small area, leading inevitably to aggregation and other forced interactions. Since one primary use of the flow FFF system is the quantitative characterization of aggregation and association phenomena, often in combination with LS and RI detectors, the reversed-flow focussing procedure is clearly a poor choice.
Another approach to solving the relaxation problem and achieving stopless relaxation is taught by Giddings in U.S. Pat. No. 5,141,651 and described by Giddings in Sep. Sci. Technol. 24(9and10), 755 (1989), both of which are incorporated by reference herein. In this method, the channel flow inlet is blocked off or occluded so as to force the incoming sample into a smaller and tighter cross-sectional distribution. When the sample-bearing flow stream encounters the crossflow stream, relaxation occurs by virtue of the existing crossflow field, so that a stop-flow period is not required. Unfortunately, this approach has also not been commercially successful, due to several inherent difficulties. One problem has been the difficulty of fabricating flow FFF devices based on the method taught by this disclosure, in that it calls for a tight seal to be provided by adjacent pieces of spacer material, usually plastic, which have a tendency to leak. Also, the solid (blocked) portion of the upper spacer component tends to bend or buckle under the force of the cross flow, at least changing the cross-sectional area of the membrane if not occluding it completely. Another problem is that reasonable resolution is not always achieved, due partly to the fact that the inlet occlusion takes up a significant fraction, up to one half, of the total channel length and also partly to the fact the channel flow stream must accelerate around the occlusion in the region of the smaller cross-sectional channel area. Yet another difficulty is that this method does not get rid of the so-called xe2x80x9cvoid peaks,xe2x80x9d which result, in part, from unrelaxed material eluting at the front of the channel flow. Given these deficiencies, it is not difficult to understand why the inlet occlusion approach has not been commercially successful.
It can be seen that each of these existing methods for accomplishing the relaxation of the analyte prior to the flow FFF separation imposes intolerable restrictions and requirements on the fractionation process, including reducing the types and variety of online detectors which can be utilized to characterize the analyte, while increasing the instrumentation complexity and operating costs, decreasing the available resolution, and rendering the respective separation processes cumbersome, inconvenient, and unnecessarily complicated to control. Since the power of flow FFF resides in the use of the correct combination of detectors, often including LS, UV, fluorescence, and RI detectors, the existing relaxation methods and devices are clearly unsuited to the application of flow FFF in many industries, including the biopharmaceutical industry.
If the remaining problem of avoiding these disadvantages of the existing relaxation procedures could be solved, while ensuring complete and efficient stopless relaxation of the incoming analyte sample and also providing a means for calculating the channel width, the flow FFF procedure would become more practical and useful, as well as more routine and automatable, which would in turn permit flow FFF to be applied to the large number of analytical and preparative problems to which it is well suited. It would therefore be highly advantageous to find a promising method for the modification of the flow FFF process to achieve stopless and splitless relaxation without any of the above-noted disadvantages.
Accordingly, the present invention is directed to an improved flow FFF process without the aforementioned deficiencies which can be implemented on commercially-available high-performance liquid chromatography (HPLC) and low-pressure liquid chromatography (LC) equipment in a manner consistent with the use of sensitive detection technologies.
The improved flow FFF process introduces the analyte into the channel in the channel-inlet flow stream (Vz), rather than in a special or dedicated flow stream, and provides an improved flow FFF process which is capable of effecting separations at enhanced rates of speed. The improved flow FFF process maintains the analyte in constant hydrodynamic motion during the separation, in order to avoid particle adsorption to any of the walls or surfaces of the channel. Relaxation is accomplished while avoiding any unnecessary steps which either artifactually concentrate or dilute the analyte. The resulting process avoids the need for stopping or reversing the axial flow during a run, and leads to dramatically-improved compatibility with flow-sensitive detectors such as LS and RI. It also minimizes the number of required pumpsxe2x80x94typically only two pumps are needed. Also avoided is the need for a channel-flow switching valve and its controlling software, and a further simplification of the remaining system plumbing, also leading to an increased ease of automation. The improved flow FFF process eliminates void peaks due to incomplete sample introduction or relaxation, and also minimizes artifactual aggregation due to concentration on the filtration membrane, so that a maximal fraction of the entire sample is included in the retained but eluted peaks. The new process and channel width calibration procedure lead to simplified and more accurate calculation of run parameters, including frictional coefficients and diffusion coefficients and their distributions, as well as improved reproducibility of retention times and calculated properties such as distributions of supramolecular masses. These and other properties render the improved flow FFF process capable of being run on commercial HPLC and LC equipment, thereby significantly extending the potential applicability of flow FFF as well as the user base of trained operators. It will be apparent to one skilled in the art that the process of the present invention is applicable to all types of flow FFF channels, including planar and annular, and to all operating modes of flow FFF, including normal, steric, and hyperlayer modes, and to all channel geometries, including parallel-walled, tapered, etc., and to flow FFF separations conducted at any temperature.
The present invention stems from a number of relevant observations. First, most if not all of the peak dispersion which is observed in stop-flow, frit inlet, and inlet splitter experiments appears to result from the way in which the sample is introduced into the channel, so that much of the excessive peak broadening has already occurred by the time that the stop-flow relaxation procedure commences. Since the sample can only undergo further broadening as it relaxes, it would seem preferable to prevent this initial broad dispersion of the analyte band rather than attempting to correct it after it has occurred, and then to minimize any additional dispersion by eliminating the stop-flow procedure. A second observation results from the recognition that the sluggishness of the field-driven transport (i.e. the cross flow rate Vx) is only relativexe2x80x94it is only slow because the channel flow Vz is typically fast, The most important and relevant metric is the linear velocity ratio Ux/Uz, which is directly proportional to retention. If the channel flow rate Vz is slowed down significantly, then the crossflow linear velocity Ux becomes relatively faster, leading to an increased linear velocity ratio Ux/Uz, in which both retention and field-driven relaxation in the cross flow are increased. Another observation is that at lower flow rates much of the momentum imparted to the entering analyte by the high linear velocity in the channel-flow inlet tubing is lost with conventional flow FFF apparatus, which employs inlet tubing of large diameters in order to accommodate the high volumetric flow rates traditionally used in flow FFF experiments.
It has been surprisingly discovered that these shortcomings can be overcome, and the foregoing operating parameters can be manipulated to obtain a new stopless and splitless method of operating a flow FFF channel which offers as good or better resolution as previous flow FFF methods, produces an analyte stream of sufficient concentration and hydraulic quality to enable detection by relevant and appropriate technologies, eliminates much of the equipment and components previously required, and renders the fractionation procedure simple, reliable, rugged, and practical. This set of conditions also avoids artifactual concentration and the need for additional dedicated software, and permit the execution of flow FFF experiments on commercially-available HPLC and LC instruments. The new method will be especially useful in the separation, characterization, and isolation of a wide variety of analytes.
The achievement of stopless and splitless flow FFF relaxation in the present invention results in fractograms which do not exhibit early-eluting system peaks, ensuring that the entire analyte sample is represented in the subsequent calculations. However, the absence of a void peak means that some other means must be found for calculating the channel width w. A useful starting point is the relationship between the retention time tr and the various experimental parameters for normal-mode flow FFF elution (M.-K. Liu et al., Prot. Sci. 2, 1520 (1993)),                               t          r                =                                            π              ·              η              ·                              w                2                            ·                              d                h                            ·                              V                x                                                    2              ·              kT              ·                              V                z                                              .                                    (        1        )            
where xcex7 is the viscosity, in poises,                               η          =                      g                          cm              ·              s                                      ,                            (        2        )            
dh is the hydrodynamic (or Stokes) diameter, k is Boltzmann""s constant,       k    =          1.38054      xc3x97              10                  -          16                    ⁢              xe2x80x83            ⁢                        g          ·                      cm            2                                                s            2                    ·          K                      ,
T is the absolute temperature in K, and Vz and Vx are the volumetric channel and cross-flow rates, respectively, in mL/min. This equation shows the direct dependence of the retention time on the hydrodynamic diameter dh, the channel width w, and the flux ratio Vx/Vz. Rearrangement of this equation yields the channel width w in terms of the difference in retention times (t2xe2x88x92t1) and the difference in hydrodynamic diameters (d2xe2x88x92d1) of two calibrant analytes,                     w        =                                                            (                                                                            t                      2                                        -                                          t                      1                                                                                                  d                      2                                        -                                          d                      1                                                                      )                            ·                              (                                                      2                    ·                    k                    ·                    T                                                        π                    ·                    η                                                  )                            ·                              (                                                      V                    z                                                        V                    x                                                  )                                              .                                    (        3        )            
where t1 and t2 are the measured retention times, in minutes, and d1 and d2 are the respective calibrating analytes"" hydrodynamic diameters, in cm.
The dependence of the retention time tr on the diffusion coefficient D can be made explicit by substituting the Stokes-Einstein equation,                               D          =                      kT                          3              ·              π              ·              η              ·                              d                h                                                    ,                            (        4        )            
into Equation 1 for the hydrodynamic diameter-based retention time t0 yield an expression for the normal-mode flow FFF retention time tr for well-retained components,                               t          r                =                                                            w                2                            ·                              V                x                                                    6              ⁢                              D                ·                                  V                  z                                                              .                                    (        5        )            
Rearrangement of this equation yields the channel width w in terms of the difference in retention times (t2xe2x88x92t1) and the difference in diffusion coefficients (D2xe2x88x92D1) of two calibrant analytes,                     w        =                              (                                          6                ·                                  (                                                            t                      2                                        -                                          t                      1                                                        )                                ·                                  (                                                            D                      2                                        -                                          D                      1                                                        )                                ·                                  V                  z                                                            V                x                                      )                                              (        6        )            
where t1 and t2 are the measured retention times, in minutes, and D1 and D2 are the respective calibrating analytes"" hydrodynamic diameters, in cm.
Another component of the present invention is the calculation of the channel width w by the use of one of these two equations or their equivalents. From the channel width w determined in this way can then be calculated the channel void time t0 and the void volume V0, as well as the numerous other parameters useful in the characterization of the analyte mentioned above, such as the distribution of diffusion coefficients or frictional coefficients, and in the characterization of the separation, such as measures of the efficiency such as the height equivalent to a theoretical plate, the resolution, the fractionating power, and the selectivity. Thus, the improved channel calibration procedure of the present invention complements the process improvements which comprise the remainder of the present invention by facilitating the calculation of the channel width w without requiring a discrete void peak in the fractogram.
The present invention comprises an improvement in the flow FFF process for the separation of particles, wherein a carrier fluid containing the particles to be separated is forced through a thin flow channel having one or more inlets and one or more outlets and a field or gradient caused by an orthogonal flow of fluid is used to induce a driving force acting across the thin dimension of the channel perpendicular to the flow axis. The apparatus for use in effecting the above-noted new process can be any conventional flow FFF channel, as described previously, and typically comprises an elongated flow channel enclosed by wall elements, a means for driving a fluid through the channel perpendicular to the long axis of the channel, an inlet means for introducing fluid into one end of the enclosed channel, an outlet means for withdrawing fluid from the other end of the channel, and adjustable flow control means for controlling the fluid flow rates being introduced or withdrawn from the channel.
The improvement of the present invention consists of 1) employing a channel spacer of a minimal thickness (preferably 762 xcexcm (0.030xe2x80x3) or less) to minimize the axial dispersion of the entering analyte sample and to maximize the high-velocity component of the relaxation process, and of 2) employing a sufficiently high crossflow rate Vx to aid in rapidly relaxing the entering analyte sample and to maximize the linear crossflow velocity ratio Ux/Uz while avoiding artifactual adhesion of the analyte to the ultrafiltration membrane, and of 3) employing as the accumulation wall an ultrafiltration membrane which is sufficiently tight-pored (preferably 1,000 kDa or less) to provide enough pressure in the channel to maintain a constant cross-sectional area throughout the axial length of the channel while also being of a size which avoids absorption of the analyte to the ultrafiltration membrane, and of 4) employing a sufficiently small channel flow rate Vz so as to minimize the linear channel flow velocity Uz to ensure the rapid and efficient relaxation of the incoming analyte sample against the ultrafiltration membrane and to maximize the linear crossflow velocity ratio Ux/Uz while maintaining a sufficiently large linear channel flow velocity Uz to ensure the presence of a Poiseuille flow velocity distribution within the channel sufficient to effect a flow FFF separation, and of 5) introducing the analyte sample in the single channel flow stream, rather than in a separate or dedicated substream, and of 6) introducing the analyte sample in a minimal volume of fluid, preferably less than about 50 xcexcL, and of 7) continuing both the channel flow and the crossflow streams uninterrupted after the introduction of the sample into the channel, and not introducing any additional substreams into the channel for the purpose of hydrodynamically relaxing the sample, and of 8) calculating the channel width w using either the formula:                     w        =                                            (                                                                    t                    2                                    -                                      t                    1                                                                                        d                    2                                    -                                      d                    1                                                              )                        ·                          (                                                2                  ·                  k                  ·                  T                                                  π                  ·                  η                                            )                        ·                          (                                                V                  z                                                  V                  x                                            )                                                          (        3        )            
or an equivalent expression for the diameter-based width or the formula                     w        =                              (                                          6                ·                                  (                                                            t                      2                                        -                                          t                      1                                                        )                                ·                                  (                                                            D                      2                                        -                                          D                      1                                                        )                                ·                                  V                  z                                                            V                x                                      )                                              (        6        )            
or an equivalent expression for the diffusion coefficient-based width, with the variables defined as shown below. Thus, the improvement of the present invention consists of the selection of a combination of eight specific experimental parameters which permit for the first time a simple and efficient technique for relaxing an analyte prior to a flow FFF separation without stopping or reversing the channel flow and without the need for additional flow substreams or complicated and expensive hardware or additional pumps. The present invention is expected to be applicable to all forms of flow FFF, including symmetrical and asymmetrical flow FFF conducted in normal, steric, and hyperlayer modes of operation in both planar and annular flow FFF.